PROJECT SUMMARY Influenza is a serious public health issue; vulnerable populations, including young children and the elderly, are especially at risk of influenza-related morbidity and mortality. Due to antigenic drift and shift of the virus as well as poor vaccine efficacy in older people, current immunization efforts fall substantially short of providing protection to the population. Research toward developing a universal influenza vaccine have been hindered by a lack of methods to model the human adaptive immune response. In this context, we have recently developed a tonsil organoid system using discarded human tonsil cells from sleep apnea patients that recapitulates at least some of the key features of an adaptive immune response against influenza, including high affinity antibodies specific for Influenza antigens and the HA molecule. We believe that this fully human system will be an ideal platform to explore and manipulate the anti-flu response in humans. In Aim 1, we will identify the minimal cellular requirements to develop protective influenza-specific T and B cell responses using these organoids. In Aim 2 we will investigate the immunomodulatory effects of adjuvants, particularly whether they influence the specificity, diversity or affinity of the influenza response. In Aim 3 we will manipulate the expression of particular genes that are likely to be important in the antibody and T cell responses and which address specific hypotheses-such as does AID play a major role in this response with respect to the specific antibodies that are generated in this system? Other genes that might alter the affinity or glycosylation pattern of the antibodies will also be investigated, as well as at least one that characterizes a uniquely flu specific response (CD38) and is expressed in germinal centers. In Aim 4 we combine computational modeling with nanoparticle and virosome stimulation of these organoids, test hypotheses about the optimal density of HA head vs stem constructs in order skew the antibody response towards broadly neutralizing, high affinity antibodies. These data could significantly aid the formulation of new vaccine strategies for the much hoped for universal flu vaccine.